JPH0794477B2 - Highly stable recombinant gamma interferon - Google Patents

Abstract

The present invention relates to novel, stable recombinant gamma interferons exhibiting in greater or less degree the antiviral and antiproliferative activity in humans and pH 2 labile properties characteristic of native human gamma interferon. The amino acid sequence of such an interferon comprises, from the N terminus, at leastwherein X is a methionine residue or hydrogen and Y is a glutamine residue or, where X is is hydrogen, Y is either a glutamine or a pyroglutamate residue.

Description

Description: FIELD OF THE INVENTION The present invention relates to the field of recombinant DNA technology, and means and methods for utilizing the above-described technology in the production of highly stable recombinant gamma interferon. It relates to production and the various products produced and their use.

BACKGROUND OF THE INVENTION For the purpose of clarifying the background of the invention and, in certain cases, supplementing details regarding the practice of the invention, numerous publications and other references are incorporated herein by reference,
For convenience, these references have been numbered and included as a list at the end of this specification. References are designated by reference numbers throughout the text.

Human interferons can be classified into three groups based on differences in antigenicity, biological properties and biochemical properties. The first group are leukocyte interferons, which are mainly produced by human blood constituent cells, which are usually virus-induced. These interferons have already been produced by microorganisms and found to be biologically active (see References 1, 2 and 3). Due to its biological properties, this interferon is intended for clinical use as a therapeutic agent for viral infections and malignant tumor conditions (4).

The second group of human fibroblast interferons is
It is usually produced by virus-induced fibroblasts. These interferons are also already produced by microorganisms and are known to exhibit a wide range of biological activities (5). Clinical trials have also suggested the potential therapeutic value of these interferons. Leukocyte interferon and fibroblast interferon have very similar biological properties, albeit with a relatively low degree of homology at the amino acid level. All of these groups of interferons are acid-stable proteins containing about 165 to about 166 amino acids.

Human gamma interferon (immunity interferon,
γ-interferon, IIF or IFN-γ) is
It exhibits antiviral and antiproliferative properties of said interferon, but is unstable at pH 2 in contrast to leukocyte interferon and fibroblast interferon. These gamma interferons were produced primarily by mitogenic induction of lymphocytes before being produced by recombinant DNA technology. Human gamma interferon is distinctly antigenically distinct from leukocyte interferon and fibroblast interferon. Gray, Goe
ddel et al. first reported the expression of recombinant gamma interferon (6), which confirmed that it exhibits antiviral activity and antiproliferative activity as well as the characteristic property of human gamma interferon, that is, instability at pH2. Was given. Gray and Goeddel produced in E. coli
Recombinant gamma interferon consists of 146 amino acids and the N-terminal part of this molecule started with the sequence CYS-TYR-CYS-. Subsequently, Derynck et al. Reported another recombinant gamma interferon with the same N-terminus and a single amino acid substitution (7), but perhaps this polypeptide was preceded by a reference (6). Allelic variant of interferon reported in
Will. Furthermore, other researchers have reported on the production of another recombinant gamma interferon, and the recombinant gamma interferon produced by them has the amino acid sequence (6) first disclosed by Gray and Goeddel. It has been reported that one or more amino acids have been replaced.

For example, Alton et al. Reported a series of IFN-s in which the 81st amino acid of gamma interferon (6) in Gray et al.
γ is reported (17), but the obtained IFN-γ is 70%.
Has only (relative basis) activity, and this IFN
-If the 1st, 2nd, and 3rd CYS-TYR-CYS of γ are deleted, the relative activity is further reduced, and IFN-γ that has only 49% activity of gamma interferon (6) such as Gray. Become.

According to the studies by the present inventors, recombinant gamma interferon containing a cysteine residue in the N-terminal amino acid sequence was
It was confirmed that the sulfhydryl groups of one or more cysteine residues pose a problem in terms of oligomerization involved in disulfide bond formation. The problem was more complex, perhaps because the reaction via the hydroxyl functional group of the tyrosine residue bound to cysteine was not possible due to the inability to completely reduce these putative disulfide bonds in our work. It seems to be included. Further, these recombinant interferons were somewhat unstable, and although it was not clear whether they were due to instability or other causes, it was found that the optimum effect could not be obtained.

SUMMARY OF THE INVENTION The present invention relates to recombinant gamma interferon having excellent stability and activity. In the most preferred embodiment, a recombinant gamma interferon having the following amino acid sequence extending from the N-terminus (hereinafter referred to as “complete sequence”), and various interferon analogs (analogs) lacking a specific site at the carboxy terminus of the sequence are described. Will be provided.

Here, X is a methionine residue or hydrogen, Y is a glutamine residue, or when X is hydrogen, a glutamine residue or a pyroglutamic acid residue. The present invention also provides corresponding cloned genes, expression vectors containing these and recombinant interferon DNA of the present invention.
Provided are transformants useful for production by the technique. The preferred recombinant gamma interferons of the invention appear to be the closest approximation to the true amino acid sequence of native gamma interferon (compared to the interferons previously described in the literature). The natural gamma interferon has been purified by the present inventors from a natural source and fully characterized. The interferons of the preferred embodiment of the present invention show significantly improved stability and activity compared to the interferons previously described in the literature.

Aspects for achieving the above and other objects of the present invention will be apparent from the following detailed description and the accompanying drawings.

FIG. 1 shows the 5'- and 3'-flanking regions consisting of untranslated DNA encoding amino acids 1-143 of the recombinant gamma interferon of the present invention and the amino acid sequence preceded by a signal sequence. Shows a DNA sequence having

FIG. 2 is a schematic diagram showing a plasmid encoding information for direct synthesis of the recombinant gamma interferon of the present invention by E. coli and a method for preparing the same.

FIG. 3 is data showing the excellent stability of gamma interferon prepared according to the present invention.

[Detailed Description] The present inventors refer to natural human gamma interferon (that is, that produced and purified by mitogen induction of human peripheral blood lymphocytes) as a recombinant gamma interferon by Gray et al. It was elucidated to be a polypeptide lacking the N-terminal CYS-TYR-CYS of the polypeptide having the sequence shown in 6). The substance obtained by digesting highly purified natural gamma interferon with trypsin according to the present inventors' research has an amino acid composition almost equivalent to the N-terminal part of recombinant gamma interferon (6) such as Gray. However, a sequence lacking CYS-TYR-CYS was contained. Since amino acid sequence analysis of native gamma interferon could not be performed from the N-terminus, the N-terminal α-amino acid of this molecule was presumed to be protected. According to Gray et al. (6), the first amino acid after the second cysteine encoded by the cDNA is GLN (glutamine), so the GLN residue is cyclized and replaced with pyroglutamic acid, resulting in a block at the N-terminus. It was supposed to have been done. Removal of pyroglutamic acid by pyroglutamate aminopeptidase liberates the next encoded amino acid, the α-amino group of ASP, allowing sequence analysis to continue, resulting in the first reported natural human gamma interferon. The same result was obtained.

By appropriately modifying the cDNA of recombinant human gamma interferon containing CYS-TYR-CYS, a protein having the above-mentioned complete sequence (where X is MET and Y is GLN) is encoded. From the cDNA it was possible to express the novel recombinant gamma interferon directly in E. coli. N-terminal methionine is the translational "start" of mRNA
It is an artificial structure encoded by the signal AUG and is not excised by the host system in the particular case of E. coli expression exemplified. In other microbial systems, such as pseudomonas, methionine can be removed. It does not appear to be necessary for activity in any case. Once methionine is removed, depending on the system used,
The GLN residue can cyclize to the form of pyroglutamic acid, but again the activity is not impaired.

According to the study of the present inventors, the recombinant gamma interferon containing CYS-TYR-CYS previously reported by Gray et al. Could be stabilized by formulating with human serum albumin. However, if serum albumin is present in the final lyophilized product, it is necessary to perform some quality control treatment prior to lyophilization rather than on the final product. On the other hand, in the case of the novel recombinant gamma interferon of the present invention, the substance in the freeze-dried state was found to be sufficiently stable without containing serum albumin. However, if desired, the gamma interferons of the invention may be formulated with pharmaceutically acceptable amounts of human serum albumin.

Furthermore, the recombinant human gamma interferon lacking CYS-TYR-CYS of the present invention appears to show more remarkable activity as an antiviral agent than the CYS-TYR-CYS-containing analog in the cytopathic inhibition test. Activity is usually assayed by inhibition of the encephalomyocarditis virus cytopathic effect (CPE) on the human lung cancer-derived cell line A549 on microtiter plates (12).

Recombinant gamma interferon of the present invention includes all interferons containing amino acids 1 to about 126 of the complete sequence. The gamma interferon with various carboxy-terminal portions cut out from the complete sequence has amino acids 1 to
As long as about 126 is present, it will still exhibit the unique properties of human gamma interferon, albeit in some cases at slightly lower levels. In fact, CYS-TYR reported in (7)
Experiments with -CYS containing analogs have shown examples where the amino acid sequence following amino acid 132 (according to the numbering of the invention) was replaced with an extraneous sequence without loss of activity (e.g. , (8)). Our preliminary data indicate that the sequence from amino acids 1 to 126 (THR) has a three-dimensional configuration that is thought to be associated with the activity relatively tightly and is associated with the activity, but the rest of the complete sequence. Amino acids support the hypothesis that amino acids are relatively structurally restricted and relatively sensitive to proteolysis. Tryptic digestion under limiting conditions removes various parts of the sequence downstream from amino acid 126 but not upstream of it. The natural gamma interferon studied by the inventors contains various molecules spanning the amino acids 127, 128, 129, 130, 132 and 134. We have found that the amino acid sequence following methionine is from amino acid 1 (after MET) 1 to about 139 and 1
We have discovered fully active recombinant gamma interferon consisting of ~ 131 different species. The latter amino acids 1 to about
131 was obtained by restriction digestion of recombinant gamma interferon with trypsin followed by sequence confirmation.
Similar tryptic digests terminating at amino acids 128 and 129 (prior to MET) retained activity, albeit substantially reduced. On the other hand, a substance having 125 amino acids (in addition to the N-terminal methionine) obtained by digesting and removing the 126th threonine and the subsequent amino acid was less than 1% of the activity of the undigested substance in the CPE inhibition assay. Only showed.

Gamma interferon induced by recombination is
In addition to having a leading methionine when the methionine is produced in the host such that it is not cleaved intracellularly, 13
It is considered that a large amount has 9 amino acids (according to the numbering in FIG. 1) and a small amount has 143 amino acids. The composition of these two species is about 9 with 139 amino acids.
Greater than 5%, most preferably greater than about 97%. Trypsin digestion under limiting conditions also removes various portions of the sequence downstream from around amino acid 126. Such restricted trypsin digests of recombinant gamma interferon according to our studies include various species spanning amino acids 125 (126 including the leading methionine), 129, 131 and 134.
These species retain their activity when stretched from around amino acids 125 to around 129, albeit substantially reduced. Species having at least approximately 129 amino acids, in particular at least 131 amino acids, ie having approximately 129-143 amino acids retain essentially and functionally full activity.

As is clear from the above, the present invention is not only the recombinant gamma interferon having the above-mentioned complete sequence, but also lacks amino acid 143 or has an amino acid sequence of Z → amino acid 143 (where Z is amino acids 127,128,129,130,131,132,
133,134,135,136,137,138,139,140,141 or 142)
It also includes various recombinant gamma interferons lacking L. and mixtures thereof. The double-stranded DNA sequence encoding the recombinant gamma interferon of the present invention encodes not only the above-mentioned DNA sequence encoding the complete sequence but also the above-mentioned various carboxy-terminal truncated analogs. It will be apparent as well to include sequences. When the various carboxy termini are truncated, the following codon encodes a translation termination signal.

In the present specification, “recombinant gamma interferon” means
By a polypeptide expressed in transformed cells from a replicable expression vehicle obtained by recombinant DNA technology, whether glycosylated or not by the cell producing it. The polypeptides of the present invention exhibit the properties peculiar to natural human gamma interferon, i.e., to a greater or lesser extent, human antiviral and antiproliferative activities, and instability at pH 2.

The recombinant gamma interferon described herein is
A "dimer" defined for the purposes of the present invention as a combination of two such polypeptides each having at least 1 to about 126 amino acids, each polypeptide having the same or a different number of amino acids. Is thought to form. Although the properties relating to the chemical bond formation mechanism have not been fully clarified, it seems to be a bond other than a covalent bond. The binding that results in dimers appears to occur spontaneously and appears to be unavoidable in the systems described herein. Therefore, when the recombinant gamma interferon of the present invention is administered, it will usually be in dimeric form.

Further, in the recombinant gamma interferon disclosed herein, the amino acid 12 is similar to the disclosure in the literature (8, 9) regarding the recombinant gamma interferon characterized separately.
Amino acid substitution or addition, particularly single amino acid substitution and multiple amino acid addition or substitution, is possible at the upstream or downstream of 6 or at the C-terminal portion without impairing the retained interferon activity. You should understand. It will be apparent to those skilled in the art that such substitutions or additions may be made without exceeding the scope of the invention. Further, the recombinant gamma interferon of the present invention includes modified species and allelic variants of the complete sequence (see FIG. 1), which have a biological activity equivalent to or higher than that of the complete sequence.

As a feature of the invention, the purified recombinant gamma interferon will be substantially free of other proteins of human origin, which distinguishes it from previously available natural human gamma interferon compositions. Will be done.

The present invention includes a recombinant gamma interferon composition of greater than about 95% purity (prior to formulation), preferably greater than about 98%, so that the composition was previously available. Distinguished from natural gamma interferon.

Similarly, one recombinant gamma interferon of an embodiment of the present invention in which the N-terminal amino acid residue is methionine is distinguished from gamma interferon produced in the human body, and is similar to the deletion of CYS-TYR-CYS. For some reason, Gray
(6) will be distinguished from the interferons having the sequence.

In the present specification, a replicable expression vehicle is a double-stranded DN.
A, preferably a plasmid, which means replication origin, promoter or promoter-operator,
It consists of a sequence coding for a ribosome binding site, a translation initiation signal codon, a gene coding for the desired recombinant gamma interferon in the proper reading phase, and a translation termination codon following the gene. Currently, recombination
The general techniques and terminology of DNA technology are well understood to those of skill in the art and, in any event, those skilled in the art will have background to the practice of all embodiments of the invention and examples that are legally equivalent. Make the necessary changes as information (1
You can refer to 1).

Example A. Cloning Messenger RN obtained from induced human peripheral blood lymphocytes as described in Reference (6) and JP-A-58-90514.
A was used to prepare a recombinant DNA clone containing the gamma interferon cDNA sequence. The DNA sequence of clone p67 is shown in FIG. Following the 5'-untranslated region, 69 nucleotides encoding a 23 amino acid precursor or signal peptide, 429 encoding a mature 143 amino acid interferon polypeptide.
5 nucleotides and forms a 3'-untranslated sequence 587
There are 4 nucleotides.

B. Expression 1. Example with E. coli In order to express recombinant IFN-γ at a high level in E. coli, the glutamine codon (amino acid 1) of the mature polypeptide was used instead of ATG (amino acid S1) of the signal peptide. The protein synthesis should start from the ATG codon immediately before () (see FIG. 1). A schematic of the procedure taken to express the p67 cDNA insert directly in E. coli is shown in FIG. A similar approach was taken to that used by Gray et al. (6) to express the cDNA insert in E. coli.

The AvaII restriction site located at codon 2 of the putative mature coding sequence was used to remove the signal peptide coding region. Two synthetic deoxyoligonucleotides were designed to regenerate the codons for amino acids 1 and 2, incorporate the ATG translation initiation codon and create an XbaI sticky end. These two oligomers are linked to the rest of the cDNA insert to encode a polypeptide of 144 amino acids and have XbaI and PstI sites at each end.
A base pair synthetic-natural hybrid gene was assembled.
This gene was cloned into XbaI and Pst of plasmid pLe IFA25 (10).
The expression plasmid pγ-CYC5 was prepared by inserting it between the I sites. This plasmid in E.coli W3110 strain (F ^-, λ ^-,
The prototrophic strain (ATCC No.27325) was transformed to obtain the host-vector system E. coli W3110 / pγ-CYC5.

2. Examples of cultured cells In order to confirm that the product is from the mature gamma interferon gene containing the N-terminal amino acid as shown in FIG. 1, both the signal peptide and gamma interferon as shown in FIG. In the presence of radiolabeled cysteine and methionine.
It was expressed in COS-7 cells (16) (unlike the case of expression by E. coli, the expression product of the mammalian cell line as exemplified here lacks the N-terminal methionine).

Confluent m layers of COS-7 cells in 60 mm Petri dishes
onolayer) using modified DEAE-dextran method to DN
Two sets of A-transfected ones were prepared. The medium was removed 3 days after the addition of DNA. 100 μCiS ^{35 on} each plate
- methionine or S ³⁵ - plus DMEM2ml added cysteine. After incubating for 16 hours in the presence of radiolabeled amino acid, the medium was removed, and the anti-gamma interferon monoclonal antibody or anti-HBsAg monoclonal antibody was used as the first antibody, and the rabbit anti-mouse IgG antibody (Cappell In
c.) was used as the second antibody to perform 500 μl of immunoprecipitation.
Reaction with the antibody and subsequent Staphylococcus (St
aphlycoccus) A cell (Calbiochem) binding to P. Ber
Described by man etc. (18). SDS-
Resuspended in mercaptoethanol and electrophoresed on a 10% SDS-PAGE gel. Gels were fixed with 7% acetic acid in ethanol, dipped in Enhance (New England Nuclear) fluorescent solution, dried and exposed for 2 weeks using Kodak AR5 film and intensifying screens (Dupont).

The plasmids used in this study were pSVγ69 (11) and pDL RI
(19) (Hepatitis B virus surface antigen expression vector, pSVγ
69 precursor) and pDLRIγ Sau (pSVγ69 (11) 830 bp
It was a polycistronic plasmid containing the Sau3A fragment, containing the entire sequence encoding gamma interferon inserted at the EcoRI site of pDLRI).
The latter plasmid contains gamma interferon and HBsAg.
It produces a transcript containing both coding regions.

S ³⁵ reacts with anti-gamma interferon antibody (A) or anti-HBsAg antibody (B) - A comparison of methionine-labeled proteins were transfected S ³⁵ in pDL RI-γ Sau - - cysteine and S ³⁵ in the cells labeled with cysteine None of the substances that specifically immunoprecipitate using anti-gamma interferon antibody migrate to the position of glycosylated (MW29,000) or monoglycosylated (MW18,000). In contrast, in contrast to this, S ³⁵ −
Immunoprecipitation of gamma interferon occurred in methionine labeled cells.

C. Fermentation production Recombinant human interferon-gamma (rIFN-γ) production using E. coli W3110 / pγ-CYC5 produced a volume of 10-1000.
Perform in batches in the l range. After fermentation, E. coli cells containing interferon were harvested from broth and rIFN-γ
Is isolated and purified. The fermentation and cell recovery methods are described below.

1) Preparation and maintenance of stock culture The following composition; Bactotryptone 10 g / L Yeast extract 5 g / L Sodium chloride 5-10 mg / L In a sterile baffled plate culture flask containing 150-500 mL of sterile medium, Prepare stock culture.

The medium is then inoculated with a primary culture of E. coli W3110 / pγ-CYC5.

The inoculated flask is then incubated at 25-37 ° C on a shaker until the absorbance at 550nm is about 1.0. 30% (V / V) dimethyl sulfoxide About 50% (V / V
V) is added to the broth. Immediately dispense a 1 mL aliquot into a sterile vial and close the lid. Store vials at -60 ° C or below. Each fermentation begins with a duplicate stock culture for inoculation.

2) Preparation of inoculum The inoculum is prepared using the above medium (LB broth) in a shake flask or small fermenter. After incubating at about 37 ° C for about 8 hours, the inoculum is transferred to the fermentor. The inoculum volume is 2-10% of the fermentation volume.

3) Fermentation The production of recombinant interferon-gamma is about 10 to 10
Performed in a fermenter with a working volume of 00l. The fermentation medium has the following composition per one:

Glucose (*) 50-100g Ammonium sulfate 4.0-8.0g Monobasic potassium phosphate 3.0-5.0g Dibasic potassium phosphate 5.0-8.0g Magnesium sulfate heptahydrate 0.5-5.1g Sodium citrate dihydrate 0.5-2.0 g UCON LB-625 0.5-2.0mL Ferric chloride hexahydrate 0.005-0.15g Zinc sulfate heptahydrate 0.001-0.15g Cobalt chloride hexahydrate 0.001-0.005g Sodium molybdate dihydrate 0.001-0.005g Ferric sulfate Dicopper pentahydrate 0.001-0.005g Boric acid 0.001-0.005g Manganese sulfate monohydrate 0.001-0.005g Hydrochloric acid 0.0-1.0mL Thiamine-HCl 0.0-0.1g Tetracycline-HCl 0.001-0.01g L-Tryptophan (*) 0.1 -0.5g Yeast extract 2.0-8.0g 3-β-indole acrylic acid 0.02-0.10g (*) Glucose and tryptophan are partly put into the fermenter first, the rest is fed during fermentation.

The components in the medium are sterilized by heat treatment or excess before being used for fermentation.

Fermentation is performed at 25-40 ° C. Other operating conditions are as follows: Agitation (rpm) 100-1000 Aeration (vvm) 0.5-1.5 (Oxygen is supplemented when necessary) pH 6.5-7.5 (Adjusted by addition of ammonium hydroxide) 4) Purification a 3.) Extraction of recombinant gamma interferon E. coli cells are suspended in a medium containing salts and a suitable buffer of pH 6 to 9, preferably about 9. Gaulin
The cell suspension is homogenized in a high pressure colloid mill such as an ulin) mill to extract recombinant gamma interferon. Add sufficient polyethyleneimine to the solution,
Prepare to a solution of 0.1 to 1% (W / V). The supernatant contains gamma interferon.

b) Partial purification of recombinant gamma interferon on silica-based adsorbents The silica-based adsorbents cleaned of impurities by washing with a suitable salt solution in the range of pH 6 to 9 are added to the above (a). Adsorb the supernatant. Elute recombinant gamma interferon with a solution containing 0.5-1.0 M tetramethylammonium chloride. All operations in this step are carried out at pH 7-9.

c) Partial purification of recombinant gamma interferon by anion exchange chromatography The eluate of (b) above is dialyzed, adsorbed on an anion exchange chromatography medium and then washed to remove impurities. Elute the recombinant gamma interferon with increasing salt gradient. Typical anion exchange resins that can be used in this step include carboxymethyl cellulose and sulfoethyl cellulose. Perform all operations on PH7-9.

d) Partial purification of recombinant gamma interferon by calcium phosphate gel chromatography The eluate of (c) above is adsorbed on a calcium phosphate medium and then washed to remove impurities. Recombinant gamma interferon is eluted with increasing salt concentration in a phosphate gradient. All operations in this step are performed at pH 7-9.

e) Partial purification of recombinant gamma interferon by anion exchange chromatography The eluate of (d) above is dialyzed, adsorbed on anion exchange chromatography medium and then washed to remove impurities. The recombinant gamma interferon is eluted from the anion exchange medium with a gradient of increasing salt concentration. Typical anion exchange chromatography media are carboxymethyl cellulose and sulfoethyl cellulose. All operations in this step are performed at pH 7-9.

f) Partial purification of recombinant gamma interferon by gel permeation chromatography The eluate of (e) above is applied to gel permeation medium and the column is developed with salt containing solvent. Appropriate fractions containing recombinant gamma interferon are pooled to obtain purified bulk. All operations in this step are carried out at pH 7-9.

g) C-terminal amino acid sequence To determine the C-terminal sequence, the sample was dialyzed into 70% formic acid, cleaved with cyanogen bromide and the resulting peptide
x Ultrasphere C8 reverse phase HPLC column separation. The peaks were collected and the amino acid and its sequence were analyzed. The C-terminal peptide detected was -LEU-PHE-ARG-GLY-ARG (residue 1
35-139, FIG. 1), and in some cases other peptides were added, and the sequence was -LEU-PHE-ARG-GLY-.
It was ARG-ARG-ALA-SER-GLN (residues 135-143, FIG. 1). To determine the proportion of these two peptides, known amounts (by amino acid analysis) were applied to a reverse phase HPLC column and the height of each peak was measured. The content of long-chain peptides (135 to 143, FIG. 1) was less than about 2% in each of the three productions, and the rest was a pentamer (having 5 amino acids). This data is from E. coli
This is consistent with the production of a mixture of 139 amino acid-containing and 143 amino acid-containing gamma interferons (without the N-terminal methionine present in each case), the relative proportion of each being approximately 98: 2%. is there.

5) Formulation The recombinant gamma interferon produced according to the above steps is made into a preferable formulation for parenteral administration in the following table.

The interferons of the invention may be used in a medically relevant dose range, for example 1.0 mg / M ² body surface area.

D. Measurement of the activity of different gamma interferons via trypsin digestion To determine the activity of different gamma interferons with different carboxy termini, gamma interferon prepared as described in the E. coli example above was used to varying degrees. It was digested with trypsin and tested by the CPE assay using A549 cells described above.

A sample (6.5 mg) of recombinant gamma interferon (r-Hu IFN-γ) prepared as described above was added to Sephadex G-25.
Desalted in 0.10M ammonium bicarbonate buffer, pH 8.5 with a molecular sieve small column (PD-10, Pharmacia),
The final protein concentration was 2.1 mg / ml. Diluted trypsin solution (Worthington TPCK trypsin, 10 μg / m in 0.001M HCl)
l, 16 μl) was added to 1.9 ml (4.0 mg) of r-Hu IFN-γ solution, mixed and incubated at room temperature (trypsin:
Protein = 1: 25,000). From the incubation mixture,
1 hour, 3.5 hours, 5.75 hours, 8 hours and 10.25
Samples were taken out after each time. 15 μl at 8th hour
(150 ng) of diluted trypsin solution was added again to facilitate the reaction for final sampling after 10.25 hours.

Bio Rad Biogel HPH
Performed on a Waters HPLC system using a T column. Aliquots of each time point in bicarbonate buffer were loaded directly onto the column (by manual injection) at the time of sampling. 0.01M
Equilibrate the column with sodium phosphate, pH 8.0, 30 μM calcium chloride and elute the protein from the column using a linear gradient of equilibration buffer and 0.5 M sodium phosphate buffer, pH 8.0, 0.6 μM calcium chloride. did.

The protein peaks detected by absorbance at 214 nm and 280 nm were collected and kept closed at 4 ° C. until used for analysis. A typical analysis for selected peaks includes:

1. Human lung cancer-derived cells Antiviral activity in A549 / EMC virus assay system (13).

2. SDS / PAG by standard method using Laemmli gel system
E-fractionation (14).

3. Determination of protein concentration by commercially available dye (Pierce Chemical Co., Rockfor, IL.) Binding method.

4. Cyanogen bromide protein cleavage followed by HPLC analysis for peptide identification (15).

The protein sample was dialyzed against 70% formic acid overnight (12,000-
(14,000 MW cutoff), dried by rotary evaporation, and resuspended in 500 μl of 70% formic acid. 12 x 7
Add solid cyanogen bromide to each sample in a 5mm glass tube,
Sealed and mixed until dissolved, covered with aluminum foil and incubated overnight at room temperature in a well-ventilated place.

After cleavage, the sample was dried by rotary evaporation, resuspended in 0.5 ml water and dried again. Prior to fractionation by HPLC, the sample was redissolved in 50% formic acid to a protein concentration of approximately 1 mg / ml.

Peptides were fractionated by a Waters HPLC system using an Altex Ultrasphere Octyl column and a trifluoroacetic acid / water-trifluoroacetic acid / acetonitrile linear elution gradient. When possible, peptides were identified by amino acid analysis. Comparative data for the truncated form of r-Hu IFN-γ are shown in Table 1 (aa represents amino acids).

It will be appreciated that any length of gamma interferon ranging from 126aa to 143aa (not including the N-terminal methionine) is expressed from a properly tailored gene. For example, the gene shown in Figure 1 is Has a Fnu4H restriction site. After the limitation by Fnu4H,
A synthetic oligonucleotide encoding the desired sequence with a "termination" codon and a linker compatible with the restriction sites available in the expression plasmid can be attached to the front of the gamma interferon gene. For example, the array: (X encodes one or more amino acids) is Fnu4H and Bgl
After digesting the plasmid with II, it can be bound to the E. coli expression vehicle exemplified above, and the termination codon resulting from the binding is as follows.

E. Assay, Cytopathic Inhibition 1. Test Method Suspension 1 of human lung cancer-derived (A549) cells (ATCC No.CCL 185) adjusted to a content of 4 × 10 ⁵ cells / ml in Eagle's MEM 1
Add 00 μl to each well.

The plates are incubated at 37 ° C for about 18 hours.

After incubating for 18-24 hours, 80 μl of medium is added to each well in the first row.

20 μl of sample for measuring interferon activity is added to the wells in the first row.

Transfer 100 μl of the contents of each well in the first row to the well in the second row next to each.

Transfer 100 μl of well contents to the next row, for a total of 10
Repeat until the 11th row.

After incubating for 24 hours, all wells except the cell control are infected with 50 μl of encephalomyocarditis virus, and the infection is repeated 24 hours after the infection so that the cytopathic effect becomes 100%.

Cover the tray with a lid and incubate at 37 ° C for 24 hours.

Drain liquid from all wells and stain with 0.5% crystal violet for 5-15 minutes.

Cell viability is measured by observing stained cells.

The sample titer is the reciprocal of the dilution at which 50% viable cells remain.

2. Calculation The activity of all samples is calculated by the following Reference
Standardize by Units Conversion Factor.

3. Specific activity A549 / EMC standardized by IFN-γ reference substance by NIH
Recombinant Human IFN Using V Bioassay System
The specific antiviral activity of -γ is about 3-fold higher than that of the modified rIFN-γ molecule having three additional amino acids (CYS-TYR-CYS) at the N-terminus.

4. Stability According to the above-mentioned measurement of biological activity (A549 / EMCV), rIFN-γ stored in the vial after preparation is stable for 3 months (stored at 4 ° C) after production (biological). There is no loss of biological activity).

F. rIFN-compared to other types of human interferon
Anti-cell proliferation activity of γ 1. Material and method rIFN-γ: CYS-TYR-CYS-deficient recombinant interferon-γ (20 mM sodium succinate, 0.15 M NaC in solution form)
l, pH 6). Prepared by the method according to the E. coli example above. The specific activity was 2.7 × 10 ⁷ IU / mg protein.

CTC-rIFN-γ: In solution form, "CYS-TY" is added to the N-terminal of the molecule.
Recombinant interferon-γ (20m with R-CYS "structure
M sodium succinate, 0.15M NaCl, pH 6). Specific activity 1.3
It was × 10 ⁷ IU / mg protein.

HuIFN-β: A lyophilized product of human fibroblast interferon produced in human diploid foreskin fibroblasts, specific activity of which is higher than 1 × 10 ⁷ IU / mg protein, and Toray Ind., Inc.
Prepared by. HuIFN-β3 × 10 ^{6 in the} vial
Contains IU and 3 mg of human serum albumin.

HuIFN-α: Dr. K. Cantell, Central Public Health Labo
Human natural leukocyte interferon in solution form provided by Ratory, Helsinki, Finland, with a specific activity of 4 × 10 ⁶ IU /
It is mg protein.

Control: Placebo containing 3 mg human serum albumin.

Culture medium: For HeLa, KB, HMV-1, FL and J-111 cells, 10% heat-inactivated pre-colostrum neonatal calf serum (PNCS) and 2 mM
Eagle's minimal essential medium supplemented with L-glutamine was used. A549 cells contain Dulbecco 'containing 10% heat-inactivated PNCS, 100 μg / ml kanamycin and 2 mM L-glutamine.
The minimum essential medium was used. RPMI 1640 medium supplemented with heat inactivated PNCS and 100 μg / ml kanamycin was used for the remaining human cells listed in Table 2.

2. Evaluation of anti-cell proliferation activity Test cells suspended in the culture medium were inoculated on a plastic tissue culture plate at a concentration of 5 × 10 ³ cells / 0.5 ml / well.
Subsequently, various amounts of interferon dissolved in the corresponding medium (0.5 ml) were added (day 0). The culture was carried out at 37 ° C. in a humid atmosphere of 5% CO ₂ and 95% air. For suspension-grown cells, the culture medium was removed on day 6 and the cells in suspension culture were directly suspended in Isoton II (Coulter Electronics Inc.) for cell counting in a Coulter counter. The cells that form the sheet in the plastic container are
Upon measurement, it was pretreated with 0.05% trypsin-0.02% EDTA to prepare a single cell suspension in Isoton II.
The anti-cell proliferative activity of interferon was 50% higher than that of control cultures (without interferon).
It was expressed as the number of antiviral units required to decrease (IC ₅₀ , IU / ml).

As shown in the table, the anti-cell proliferative activity of rIFN-γ was significantly different among human cell types. In this case, KATO-III, a gastric cancer siglet-ring cell, is highly sensitive and has an IC of 1.2 IU / ml.
₅₀ shows that Daudi cells, that is, Burkitt lymphoma, are highly sensitive to type I interferons (HuIFN-α, HuIFN-β), whereas they are not sensitive to type II interferons including rIFN-γ. It was sensitive. Lung adenocarcinoma (PC
-8, PC-12) was insensitive to all interferons tested. The anti-cell spectra between rIFN-γ and CTC-rIFN-γ are almost identical, but generally rIFN-γ
The anti-cell proliferative effect of γ seems to be superior to that of CTC-rIFN-γ. When comparing the four interferons, the highest effect was obtained on rIFN-γ except for Daudi cells.

G. In vitro rIFN-γ and CTC-rIFN in various liquids
-Comparison of gamma stability 10 mg human serum albumin, phosphate buffer and isotonic Na
Freeze-dried product of rIFN-γ prepared according to the example of E. coli, each containing Cl, of 1 × 10 ⁶ IU / vial and rIFN-γ having “CYS-TYR-CYS” structure at the N-terminus. Dissolve 1 × 10 ⁶ IU / vial of lyophilized product with distilled water to make the concentration 4
It was adjusted to × 10 ⁴ IU / ml.

The in vitro stability of interferon was evaluated by measuring the residual antiviral activity in various liquids. The above interferon solution was pre-incubated for 10 minutes in a water bath incubator at 37 ° C or 4 ° C, and then 9 volumes of rabbit serum, human serum or Eagle
Incubation was initiated by adding to e'sMEM. Aliquots were taken after 0,0.25,0.5,1,4,8,24,72 and 144 hours and mixed with 9 volumes of Eagle's MEM. The sample was frozen and stored at -80 ° C in a deep freezer until the interferon titer was measured. Interferon titer was measured by the CPE ₅₀ method using human amnion cells (FL cells) infected with Sindbis virus. The results are shown in FIG. 3 as the percentage of residual titer relative to the initial titer.

Although the invention has been illustrated with reference to the preferred embodiments expressed in E. coli and COS-7 cells, the recombinant gamma interferon of the invention may be used in other bacterial strains, yeast and other systems such as tissue culture systems. It is clear that it can also be produced in. See JP-A-58-90514 and reference (6).
That is, the invention is not limited to the most preferred embodiments, but rather to all legally equivalents of the appended claims.

References 1.D.Goeddel et al., Nature, 287 , 411 (1980) 2.D.Goeddel et al., Nature, 290 , 20 (1981) 3.E.Yelverton et al., Nucleic Acids Research, 9 , 731
(1981) 4.Gutterman et al., Annals of Int. Med. 93 , 399 (198
0) 5.D.Goeddel et al., Nucleic Acids Research, 8, 4057
(1980) 6.P.Gray et al., Nature, 295 , 503-508 (1982) 7.R.Derynck et al., Nucleic Acids Research, 10 , 360
5 (1982) 8.R. Derynck et al., Interferon Scientific Memorand
a, August 1982, Memo-I-A1193 / 2 9.R.Derynck et al., “Expression of Human Interfero
n Gamma in Heterologous Systems "in Experimental Ma
nipulation of Gene Expression, Academic Press, Inc.2
47 (1983) 10.D.Goeddel et al., Nature, 287 , 411-416 (1980) 11.D.Goeddel et al., European Patent Application Publication No. 0,077,670 12.WEStewart II, “The Interferon System”, Springe
r Verlag (New York) pp.13-26 (1979) 13.Stewart, “Interferon Systems” ′ Ed.:Stewart, p.1
3, Springer-Derlag, New Yok (1979) 14.Laemmli, Nature, 227,680 (1970) 15.Gross, et al., Methods in Enzymology, XI, 238 16.Gluzman, Cell, 23,175 (1981) 17.Alton et al., “Production, Characterization and
Biological Effects of Recombinant DNA Derived Huma
n IFN−alpha and IFN−gamma Analogs ", The Biology o
f the Interferon System, De Maeyer and Schellekens,
Eds., Elselvier Science Publ. (1983). 18.Berman et al., Science, 222 , 524 (1983) 19.Simonsen et al., Mol, Cell.Biol 3 , 2250 (1983)

[Brief description of drawings]

FIG. 1 is a diagram showing the nucleotide sequence of the recombinant gamma interferon gene of the present invention and the amino acid sequence deduced therefrom, and FIG. 2 is a plasmid encoding the recombinant gamma interferon directly synthesized by the present invention. FIG. 3 is an explanatory view showing the preparation method of, and FIG. 3 is a graph showing the excellent stability of the gamma interferon of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical display location C12R 1:19) (C12P 21/02 C12R 1:91) (56) Reference JP-A-58- 90514 (JP, A) JP 59-51792 (JP, A) International Publication 83/04053 (WO, A) Nature, 295 (1982) P. 503- 508 Edward De Maeyer, H uub Sehellekens ed., "Th e Biology of the In terferon System 1983- Proceeding of the S econd Interrnationa l TNOMeeting on the Biology of the Int erferon System, held in Rotterdam, The N etherland on 18-22 Apr. ”(1983) P. 121-128

Claims (2)

[Claims] 1. The following amino acid sequence extending from the N-terminus: [Wherein X represents a methionine residue and Y represents a glutamine residue, or X represents hydrogen and Y represents a glutamine residue or a pyroglutamine residue]. peptide. 2. The following amino acid sequence extending from the N-terminus: [Wherein X represents a methionine residue and Y represents a glutamine residue, or X represents hydrogen and Y represents a glutamine residue or a pyroglutamine residue]. Peptide and the following amino acid sequence extending from the N-terminus: [Wherein X represents a methionine residue and Y represents a glutamine residue, or X represents hydrogen and Y represents a glutamine residue or a pyroglutamine residue]. A composition containing a peptide, wherein the former content is 95% or more.